Coding

Part:BBa_K2023009

Designed by: Clia Chenebault   Group: iGEM16_Ionis_Paris   (2016-10-14)


Gaussia luciferase coding sequence optimized for use in E.coli DH5-Alpha

This biobrick is the GLuc-His (BBa_K1732027) that we optimized for E.Coli DH5-Alpha and for the IDT sequence order.

GLuc-His gene is found in a well-known organism, the copepod Gaussia princeps. It encodes for the Gaussia luciferase (followed by 6 histidines) enzyme, also known as GLuc, which is involved in a bioluminescence process. This enzyme degrades coelenterazine, into a product, celenteramide. With an optimal substrate level, this step produces energy in the form of light that can be detected with a fixed spectrophotometer at 488nm.
The Gaussia luciferase is an ideal reporter gene because of its stability at high temperature thanks to disulfide bonds and because it has extremely high activity in light production for very sensitive assays. When compared to Firefly and Renilla luciferase, GLuc generates over 1000-fold higher bioluminescent signal intensity[1][2]. The NanoLuc has an activity a little higher but this luciferase is very recent and thus less characterized. Advantages of luminescence, over fluorescence, include the absence of background noise, the amplification of signal and a high dynamic range that spans many orders of magnitude. Indeed, since light emission depends strictly on the chemical reaction between the substrate and the luciferase, there is no background noise originating from the sample [3]. Furthermore, the turnover of the light reaction significantly amplifies the reporter signal. Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis. Even though bioluminescence is currently used mainly for transcription study and cell imaging, this method become increasingly popular for quantitative analysis. [3]

[1] Inouye, S., Sahara-Miura, Y., Sato, J., Iimori, R., Yoshida, S., and Hosoya, T. (2013). Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: Comparison of substrate specificity for C2-modified coelenterazines. Protein Expression and Purification 88, 150–156.
[2] S. Inouye, Y. Sahara, Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps, Biochem. Biophys. Res. Commun. 365 (2008) 96–101
[3]Wood, K (2011), The bioluminescence advantage, laboratorynews, available on: http://www.labnews.co.uk/features/the-bioluminescence-advantage-13-09-2011/

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 94
    Illegal NheI site found at 505
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//function/reporter/light
Parameters
proteinGaussia Luciferase